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Available online at www.sciencedirect.com

Separation and Purification Technology 61 (2008) 243–248

Contribution of pH dependent osmotic pressure to amino acid transportthrough nanofiltration membranes

Zoltan Kovacs ∗, Wolfgang SamhaberInstitut fur Verfahrenstechnik, Johannes Kepler Universitat Linz, Welser Straße 42, A-4060 Leonding, Austria

Received 22 June 2007; received in revised form 29 October 2007; accepted 30 October 2007

bstract

A concept is presented to determine the osmotic pressure of amino acid solutions by altering the stoichiometric coefficient of the van’t Hoff laworresponding to the dissociation state of the amino acids. The osmotic pressure is greatly affected by the feed pH, which significance has beenverseen in membrane permeation studies. Nanofiltration experiments were carried out in order to investigate the effect of osmotic pressure on theermeation of amino acids through polymeric membrane. l-Alanine aqueous solutions of various feed pH (6.0, 9.7 and 11.1) were concentrated

n discontinuous operation mode with complete retentate recycle. It has been stated by many authors, that polymeric nanofiltration membranesossess higher rejection for amino acids in negatively charged form than for zwitterions. In our study we demonstrate that this tendency can beeversed in higher concentration range.

2007 Elsevier B.V. All rights reserved.

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eywords: Nanofiltration; Amino acid; Concentration; Osmotic pressure; pH

. Introduction

Nanofiltration (NF) is a relatively new class of pressure-riven membrane techniques that lies between ultrafiltration andeverse osmosis. NF is operated at medium pressures in the rangef 5–50 bar. Both size and charge of solute play an important rolen the separation behaviour of NF membranes. The transporta-ion of non-charged solutes through an NF membrane is usuallyharacterized by the term of molecular weight cut-off, which isn the range of about 200–1000 Da. Polymeric NF membranesre known to have considerably higher rejection for ionic specieshan for neutral solutes of the same molecular weight due to theroperties of electrostatic repulsion as well as size exclusion.

NF membrane processes have found their way into indus-rial applications of various fields as viable alternatives to moreraditional separation processes like extraction, ion-exchange,vaporation and distillation. In the synthesis and down-streamrocessing of amino acids (AA), the purification and recovery

s a challenge, where NF is a promising separation tool. Theotential use of NF for such purposes has been recently studiednd discussed by several authors, which had covered the field

∗ Corresponding author. Tel.: +43 70 672509x21; fax: +43 70 6725095E-mail address: zoltan.kovacs@jku.at (Z. Kovacs).

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383-5866/$ – see front matter © 2007 Elsevier B.V. All rights reserved.oi:10.1016/j.seppur.2007.10.018

f enzymatic glutathion synthesis [1], separation of glutaminerom fermentation broth [2], aspartame production [3], frac-ionation of protein hydrolysates [4–6], cofactor regenerationn l-tert-leucine enzymatic synthesis [7].

Amino acids are ionisable compounds. In neutral aque-us media, diprotic AA with non-ionisable residual group areredominantly present in zwitterionic form as a result of inter-olecular proton transfer. If the pH of the solution is higher than

heir isoelectric point, dissociation takes place, which resultshe anionic form. It has been reported in several studies, that

considerably higher rejection can be observed for AA ofnionic form than for zwitterions [1,3,8,9]. It should be noted,hat these studies have been focused on highly diluted sys-ems of 10−2 to 10−3 M feed concentration range. Wang et al.imulated NF process for concentration and diafiltration of l-henylalanine/l-aspartic acid solution based on rejection datachieved from laboratory measurements using highly dilutedystems [3]. Fairly less experimental data are available on con-entrated solutions. Timmer et al. [10] demonstrated the effect of.1 M feed concentration on NF and also Li et al. [2] studied theejection of l-glutamine and l-glutamic acid single solutions

ith the variation of feed concentration up to their solubil-

ty limit. It was concluded, that the rejection of charged AAs decreasing much more with increasing feed concentrationhan in the case of AA of zwitterionic form. Such a finding

2 d Purification Technology 61 (2008) 243–248

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44 Z. Kovacs, W. Samhaber / Separation an

as interpreted by the authors as a result of a change in theharged nature of the membranes. Namely, the saturation of theharged sites on the membrane is claimed by the authors to pro-oke a more permeable medium for charged components dueo charge shielding. Estimation and use of effective membraneharge density for practical applications is rather complex ando quantitative description of the transport phenomena of aminocids has been reported so far. For the sake of completeness weave to mention, that an increase in the rejection of l-glutamineith increasing feed concentration was found by Li et al. [2].his unusual behaviour has been explained by the authors as

he result of association of l-glutamine into dimers and poly-ers. On the basis of the current state of research, no definite

onclusion on the concentration dependency of rejection can berawn. Since NF is a pressure driven process, information on thesmotic pressure of the feed is essential. The pH dependency ofhe osmotic pressure exerted by AA solutions and the impact ofhis phenomenon on the separation behaviour seem to be left outf consideration in the literature yet.

In the following, we report the concept for calculating thesmotic pressure of diprotic amino acid solutions highlightinghe role of feed pH and discuss the impact of it on membraneermeation behaviour based on experimental results.

The following discourse will deal with the case of alaninend its permeation studies through an NF membrane, whichill, however, be sufficient to indicate the general nature of the

elevant phenomena.

. Theory

.1. Osmotic pressure of amino acid solutions

There is a lack of available information on osmotic pressureata of amino acid solutions in the open literature, although its expected to be of special significance in several disciplines ofcience.

The dissociated species of a diprotic amino acid in aqueousolution occur in equilibrium as follows:

OOC–R–NH3+ � −OOC–R–NH3

+ � −OOC–R–NH2

e consider a cation fraction fA+ , an anion fraction fA− and awitterionic fraction fA0 of the total amino acid concentration.he equilibrium can be expressed with K1 and K2 dissociationonstants:

= fA+ + fA− + fA0 (1)

1 = cH+cA0

cA+(2)

2 = cH+cA−

cA0(3)

sing the identities of pK1 = − log K1, pK2 = − log K2, pH =

log cH+ , and combining with (1)–(3), the following relations

an be delivered:

A+ = 1

1 + 10pH−pK1 + 102pH−pK1−pK2, (4)

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ig. 1. Estimated osmotic pressure in the function of pH for alanine solutionsf different concentrations at 25 ◦C.

A− = 1

1 + 10pK2−pH + 10pK1+pK2−2pH (5)

he osmotic pressure can be determined by van’t Hoff law:

= iRTcA (6)

he factor i is evidently equal to the sum of the number ofnactive molecules, plus the number of ions, divided by the sumf the inactive and active molecules [11]. For a diprotic aminocid this gives

= fA0 + 2fA+ + 2fA−

fA0 + fA+ + fA−(7)

hich can be converted into the equivalent form of i = 1 +A+ + fA− . Combining (4)–(6) results in

=[

1 + 1

1 + 10pH−pK1 + 102pH−pK1−pK2

+ 1

1 + 10pK2−pH + 10pK1+pK2−2pH

]RTcA (8)

q. (8) expresses the pH dependency of the osmotic pressure ofdiprotic amino acid, which is graphically illustrated in Fig. 1.

The net charge z is given by z = (+1)fA+ + (−1)fA− +0)fA0 . Fig. 1 shows that complete ionisation (z = +1 or −1)esults a twofold increase in the osmotic pressure compared withhe non-charged state (z = 0). This difference in the osmoticressure can cause remarkable consequences on the permeationhrough the membrane especially in higher concentration ranges,here the exerted osmotic pressure of the feed is in the samerder of magnitude with the applied pressure. Compare withhe generally applied 10–40 bar pressure in NF, considering fornstance a 1 mol/L solution, the exerted osmotic pressure for

eutral and alkaline regime is ca. 25 and 50 bar. Since the solu-ion property is expressed in term of pressure, it can be used inractical calculations and readily applied in predictive models.

Z. Kovacs, W. Samhaber / Separation and Purification Technology 61 (2008) 243–248 245

Table 1Characteristics of alanine

Item Alanine

Symbol AlaMolecular formula C3H7NO2

Molecular weight (g/mol) 89.09pK, �-COOH group 2.34pK, �-NH3

+group 9.69pS

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.2. Permeability equations

Many models have been proposed for NF so far, which can beivided into two main types: transport mechanism models andrreversible thermodynamics models (IT).

The models based on phenomenological relationships of thehermodynamics of irreversible processes have been widely usedn predicting transport through NF membranes for single andinary solute systems [12,13], most recently for multiple sys-ems [14] and also for industrial feeds [3,15–17].

The set of basic equations is given by Kedem and KatchalskyKK) for jv total volume flux and js molar solute flux [18]:

v = Lp(�P − σ�π), (9)

s = ω�π + (1 − σ)Jvc, (10)

here Lp, ω and σ are membrane transport coefficients, c theean solute concentration within the membrane, �P the trans-embrane pressure and �π is the osmotic pressure difference.In non-equilibrium thermodynamics, the membrane is treated

s a “black box”, so no insight into the transport mechanismss provided. The KK model neglects the effect of membraneharge. It is generally true, that polymeric NF membranes haveigher rejection for negatively charged than for zwitterionicmino acids in diluted systems. The reason of this differencen the solute permeability in such diluted systems, as well as thenner mechanism of the membrane effect itself would require a

ore complex theoretical description, which will at this stagee left aside. Our objective is to demonstrate the effect of thesmotic pressure. According to our prior hypothesis, the osmoticressure as a function of ionization can be described by Eq.8). In diluted systems and at adequately high transmembraneressure, where σ�π � �P , the osmotic pressure differenceetween charged and uncharged solutes is less pronounced. Inontrary, at higher concentration range, the pH dependency ofhe osmotic pressure can result a significant change in the volumeux and has a greater overall influence on solute permeabilityccording to Eqs. (9) and (10).

. Experimental

.1. Materials

l-Alanine of analytical degree used in the experiments wasurchased from Merck. The properties are shown in Table 1.

dott

Fig. 2. Schematic diagram of the lab-scale membrane system.

Commercial polymeric DK nanofiltration membrane is usedn this study. This thin film composite membrane was suppliedy GE W&P Technologies, characterized by an approximateolecular weight cut-off of 150–300 Da.Concentration of Ala was determined in pre-diluted solutions

sing a total organic carbon analyzer (TOC 500, Shimadzu).djustment of pH was made with a low ionic double junctionH electrode by adding 5 M NaOH. Osmotic pressure of Alaolutions was determined by vapour pressure osmometry withnauer K-7000 osmometer using standard NaCl solutions ofnown osmolalities for calibration.

.2. Experimental set-up

Nanofiltration was performed using the experimental set-uphown in Fig. 2.

The membrane unit is equipped with a LAB20 typelate-and-frame module manufactured by DDS (De Danskeukkerfabrikker, Denmark). Two flat-sheet discs of DK mem-rane with the effective membrane surface area of 350 cm2werelaced in the module. Retentate stream was recycled to the feedank and permeate was collected in a separate vessel duringhe process. The circulating velocity was set to 500 L/h, whichs the recommended value by the manufacturer, as necessaryor restrained concentration polarization, corresponding to aeynolds number of 965 [19]. Applied trans-membrane pres-

ure was adjusted to 30 bar with a pressure valve at the retentateide. All experiments were carried out at 25 ± 0.2 ◦C.

.3. Methods

The experimental work was carried out in three batches, allf them following the same procedure and under the same con-

itions but at different feed pH. First, an aqueous 0.2 M solutionf Ala was fed in the feed tank and concentrated in discon-inuous operation mode with complete retentate recycle. Theotal volume of the permeate was measured in order to cal-

2 d Purification Technology 61 (2008) 243–248

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Fig. 3. Rejection in the function of concentration factor for the different batches.

Fb

tctstage of the process. This phenomenon is shown in Fig. 5.

Rejection of zwitterionic form decreased from 72 to 63%,whereas, on the other hand, the rejection drop for anionic formwas from 89 to 54%. Such behaviour has been interpreted by the

46 Z. Kovacs, W. Samhaber / Separation an

ulate the volumetric concentration factor of the feed solutionn = Vfeed/Vconcentrate). During the process, permeate and reten-ate samples were periodically taken from the permeate pipend from the feed tank, respectively. Ala rejection was thenalculated using the formula:

= 1 − cp

cr, (11)

here cp and cr are the solute concentrations of permeate andetentate, respectively. The permeate flux was manually mea-ured by using a calibrated cylinder and a stopwatch. Volume ofollected permeate and operation time was recorded. The con-entration process was stopped, when the Ala concentration ofpproximately 0.9 mol/L had been achieved. Then the collectedermeate was recycled and mixed again with the concentraten the feed tank. Thereafter, the pH was adjusted to 9.7, whichorresponds to 50% dissociation, and the concentration processas repeated in the same way than as was before at neutral pH.inally, in the third batch, the operation was carried out by set-

ing the pH to 11.1, which results a close to complete ionisationf the Ala.

After the experimental part, deionised water permeability ofhe membrane was checked and complete recovery of the initialermeability was verified.

. Results and discussion

Results of the batch-mode NF experiments of pH 6.0, 9.7 and1.1 are summarised in Table 2. The initial feed solutions wereoncentrated up to about 0.9 mol/L, which required 4.7, 5.7 and.3 h of operation time for the respective batches.

Rejection and flux data plotted against the concentration fac-or are shown in Figs. 3 and 4.

At low concentration range, an increase in the ionisationegree results increasing rejection. Initial rejections were 72,8 and 89% at pH 6.0, 9.7 and 11.1, respectively. This ten-ency is in a good agreement with literature data of dilutediprotic amino acid solutions [1–3,8] and in particular, con-rms the observations on permeability of �-Alanine throughellulose acetate membranes demonstrated by Matsuura and

ourirajan [9]. While the rejection of zwitterionic Ala decreasesnly slightly (with a total of 12%) during the process, a substan-ially larger drop can be observed in the batches where Ala hashe net charge of z = −0.5 (27%) or z = −1 (39%). The rejec-

able 2ummary of batch NF operations carried out at different pH values

pH

6.0 9.7 11.1

peration time (h) 4.7 5.7 9.3oncentration factor 7.5 7.1 5.5inal concentration (mol/L) 0.89 0.90 0.86lux decrease during operation (%) a 58 72 81ejection decrease during operation (%) b 12 27 39

a Defined as (1 − Jfinal/Jinitial) × 100%.b Defined as (1 − Rfinal/Rinitial) × 100%.

Fb

ig. 4. Permeate flux in the function of concentration factor for the differentatches.

ion of charged Ala molecules decreases by increasing retentateoncentration and not only reaches the rejection value of zwit-erionic form, but even lower values were measured at the final

ig. 5. Rejection in the function of retentate concentration for the differentatches.

Z. Kovacs, W. Samhaber / Separation and Purification Technology 61 (2008) 243–248 247

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atiedhighly concentrated solutions can result lower observed rejec-tion for anionic than for zwitterionic solutes of the same feedconcentration.

ig. 6. Estimated osmotic pressure (Eq. 8) vs. Ala concentration curves withhe experimental results of vapour pressure osmometry (at 25 ◦C).

aturation of the charged sites of NF membranes. This saturationesults weaker electrostatic repulsion and it is claimed to explain

large rejection drop for charged solutes by increasing feedoncentration, but so far, no sufficient explanation was reportedn why charged AA is more permeable in comparison with non-harged AA at high feed concentration regime.

Rejection cannot be discussed independently of flux data. Ast is shown in Fig. 4, high flux differences between the batchesere found even at the process begin, although these flux data

efer to the same, 0.2 mol/L Ala feed concentrations. The initialermeate fluxes were 92, 83 and 64 L/(h m2) for the batchesH 6.0, 9.7 and 11.1, respectively. These differences can bexplained by taking into account the osmotic pressure of the feedolutions. Increasing degree of ionisation results an increase inhe osmotic pressure as it is shown in Fig. 6.

At the initial feed concentration, it is approximately 5 baror zwitterionic, 7.5 bar for half-dissociated and about 10 baror practically completely dissociated molecules. The experi-ental values measured by vapour pressure osmometry are ingood agreement with the estimated values. Hereby we have

o remark, that because of amino acids act as buffers, a smallnaccuracy in adjusting the feed pH to the pK value of Ala canause a relatively great difference in the exerted osmotic pressurealues, which can gain major importance in membrane processontrol. Stable, less pH dependent osmotic pressure values cane adjusted around the isoelectric point and at the dissociationevel of close to unity. The osmotic pressure of the solution stillould be raised after all Ala molecules are converted into anionicorm by further addition of NaOH. In this case, no extra osmoticressure gradient across the membrane would be built, since theembrane is virtually freely permeable for ionic species pro-

uced by the dissociation of the excessive NaOH. The permeateux decreases during the concentration process. As it is shown

n Fig. 7, the relative decrease of flux (i.e., Jactual/Jinitial ×00%) within the process was found to be unequal for the

atches.

The greatest decrease (81%) of the initial flux by the end ofhe process was achieved in batch pH 11.1, in contrary, medium72%) for pH 9.7, and low (58%) for pH 6.0 were measured. F

ig. 7. Relative flux decrease in percentage defined as Jactual/Jinitial × 100%s. retentate concentration for the different batches.

onsidering a final concentration of 0.9 mol/L on the feed sideor osmotic pressure calculation, it results about 22, 33 and 44ar for net charge of z = 0, −0.5 and −1, respectively. Thesealues are of the order of the applied transmembrane pressure,hich is 30 bar. It is to be noted, that for a completely reject-

ng membrane, in the case of completely dissociated moleculese would require more than 44 bar external pressure to gainermeate flux as it is the basis of reverse osmosis. Based onq. (9) we see that positive flux for a partially retentive NFembrane is the result of the lower osmotic pressure gradi-

nt across the membrane (�π = Rπfeed, with R < 1) and ofhe convective interaction between solute and solvent (σ < 1).he rejection in the function of permeate flux is shown inig. 8.

One can clearly see, that a decreasing permeate flux resultsdecrease in the rejection. Since the anionic Ala exerts twofold

he osmotic pressure of the zwitterionic Ala, this differencen the feed side can be identified as a major factor for thextensively pronounced permeate flux decrease. This enhancedrop in the flux between the differently charged solutes in

ig. 8. Rejection in the function of permeate flux for the different batches.

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48 Z. Kovacs, W. Samhaber / Separation an

. Conclusions

1) A quantitative and simple theory of pH dependence of theosmotic pressure exerted by diprotic amino acids is pre-sented.

2) The pH dependency of osmotic pressure was verified exper-imentally by vapour pressure osmometry.

3) The significance of such phenomenon in membrane trans-port is pointed out.

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[

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